1. Field of the Invention
The present invention relates generally to nitric oxide-releasing molecules. More specifically, the present invention relates to carbon-based diazeniumdiolate nitric oxide-releasing molecules.
2. Background of the Invention
Nitric oxide (NO) is a bioregulatory molecule with diverse functional roles in cardiovascular homeostasis, neurotransmission and immune response (Moncada et al., 1990; Marletta et al., 1990). Because NO influences such a vast array of physiological activity, it is desirable to have compounds release NO for use as drugs and physiological and pharmacological research tools. Even more desirable are non-toxic, non-carcinogenic compounds that can generate NO under physiological conditions for therapeutic and clinical applications. Such compounds, however, have been difficult to develop.
Small molecules (generally described as molecules with Formula Weights less than 600) that release NO are well known, and some classes such as the organic nitrates have been used for decades therapeutically.
Diazeniumdiolates are a class of compounds which contain the —[N(O)NO]— functional group and have been known for over 100 years (Traube, 1898). Molecules that bear the diazeniumdiolate group have been disclosed as NO-releasing agents (U.S. Pat. Nos. 4,954,526; 5,039,705; 5,155,137; and 5,208,233). Here the diazeniumdiolate is attached to a primary amine, secondary amine or a polyamine and spontaneously produce nitric oxide under physiological conditions. An advantage to these NO-releasing agents is their wide range of half-lives depending upon the structure of the amine bearing the diazeniumdiolate group (Keefer et al., 1996). The major disadvantage associated with these nitrogen-based diazeniumdiolates is the potential formation of carcinogens (nitrosamines) upon decomposition and release of NO as shown in Equation 1 (Parzuchowski et al., 2002). Some nitrosamines are highly carcinogenic and the potential for nitrosamine formation limits the N-based diazeniumdiolate class of NO donors from consideration as therapeutic agents based on safety issues.

Other non-diazeniumdiolate forms of NO donors have been described including S-nitroso compounds (U.S. Pat. Nos. 5,536,723, Loscalzo et al. and 5,574,068, Stamler et al.) and C-nitroso compounds (U.S. Pat. No. 6,359,182, Stamler et al.). Regarding the S-nitroso compounds, their therapeutic potential is limited due to their rapid and unpredictable decomposition (release of NO) in the presence of trace levels of Cu(I) and possibly Cu(II) ions (Dicks et al., 1996; Al-Sa'doni et al., 1997). Furthermore, S-nitroso compounds may decompose by direct transfer of NO to reduced tissue thiols (Meyer et al., 1994; Liu et al., 1998). Finally, many mammalian enzymes may catalyze the release of NO from S-nitroso compounds (Jourd”heuil et al, 1999a; Jourd”heuil et al., 1999b; Askew et al., 1995; Gordge et al., 1996; Freedman et al., 1995; Zai et al., 1999; Trujillo et al., 1998). However it is well known that tissue and blood levels of ions, enzymes, and thiols are subject to a wide range of variability in each individual, thus making the release of NO unpredictable from subject to subject. The dependence and sensitivity of NO release on blood and tissue components limits the therapeutic potential of nitroso compounds in medicine.
Several references to carbon- or C-based diazeniumdiolate molecules which release NO have been disclosed (U.S. Pat. Nos. 6,232,336; 6,511,991; 6,673,338; Arnold et al. 2000; Arnold et al. 2002; Arnold et al. 2002). C-based diazeniumdiolates are desirable because in contrast to N-based diazeniumdiolates they are structurally unable to form nitrosamines while maintaining their ability to spontaneously release NO under physiological conditions. Hrabie et al. describe a series of enamine-derived diazeniumdiolates of which only one spontaneously releases a small amount of NO (approximately 7% of the theoretical maximum) under physiological conditions (Hrabie et al., 2000; U.S. Pat. No. 6,232,336).
Furthermore, there have been recently published reports on NO-releasing imidates, methanetrisdiazeniumdiolate, and a bisdiazeniumdiolate derived from 1,4-benzoquinone dioxime (Arnold et al. 2000; Arnold et al. 2002a; Arnold et al. 2002b). However the dioxime, which had a favorable NO-release profile of 2 moles of NO per mole of compound, breaks down to a carcinogen (Westmoreland et al., 1992). The methanetris compound is explosive (Arnold et al., 2002) and the imidate class of compounds can cross-link proteins (discussed in detail below).
Arnold et al. disclose NO-releasing imidates and thioimidates of the following general structure (I) (U.S. Pat. No. 6,673,338):
They also disclose embodiments where the imidate functional group is used to covalently bind the molecule to polymers or biopolymers (proteins), as the imidate functional group is commonly used to bind and/or cross-link proteins (Sekhar et al., 1991; Ahmadi and Speakman, 1978). However the protein binding properties of imidates may be undesirable in applications involving contact with blood, plasma, cells, or tissue because the imidate may react with tissue protein.
Thus there continues to be a need for NO-releasing molecules that release NO spontaneously under physiological conditions and in predictable and tunable quantities of NO, where the NO release is not affected by metals, thiols, enzymes, or other tissue factors that may result in variable NO release, and where the molecule cannot decompose to form nitrosamines and does not covalently bind proteins.
Therefore, it is an object of the present invention to provide a composition that includes a C-based diazeniumdiolate that can generate fluxes of NO spontaneously under physiological conditions. It is a further object of the present invention to provide NO-releasing molecules that generate predictable and tunable NO release rates. It is a further object of the present invention to provide diazeniumdiolate molecules that do not decompose into nitrosamines or covalently bind proteins.
In addition, it is an object of the present invention to provide a method of synthesis for the C-based diazeniumdiolates molecule. A further object of the present invention is to provide methods of use for the C-based diazeniumdiolate molecules in biology and medicine. Further objects and advantages of the invention will become apparent from the following descriptions.